Molecular diagnosis and prognosis of carcinomas

ABSTRACT

The present invention is directed to the use of human BC200 RNA in both the diagnosis and prognosis of carcinomas to determine the presence of a carcinoma and tumor grade, and also to predict the likelihood that a carcinoma will progress to an invasive carcinoma.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase application of PCT/US03/05502,having an international filing date of Feb. 21, 2003, which claimspriority to U.S. Provisional Application No. 60/359,156, filed Feb. 22,2002, the contents of which are incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OF DEVELOPMENT

This invention was made with Government support under DOD Grants DAMD17-96-1-6201 and DAMD 17-02-1-0520. The Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to the use of a molecular marker, BC200 RNA, inscreening for neoplastic diseases. Methods may be used by which BC200RNA expression may be monitored and utilized for both the diagnosis andprognosis of carcinomas.

2. Background of Related Art

Progress in the diagnosis and prognosis of cancer has been hampered bythe lack of suitable, reliable and sensitive molecular markers. Suchindicators are necessary to identify lesions and characterize them, todistinguish benign from malignant tumors, and to be able to determinewhether a given non-invasive carcinoma will become invasive in thefuture.

For example, no reliable molecular marker is currently available thatcould be used to complement mammography in the detection of breastcancer. This contrasts with prostate cancer, for example, whereprostate-specific antigen (PSA) status can be established by simplyanalyzing a blood sample. PSA is a molecular marker for prostate cancer(reviewed by Gamick et al., “Combating Prostate Cancer”, Sci. Am.279:75-83 (1998)), and although PSA status is not a very reliable tumorindicator (with relatively high false negative and false positiverates), it is routinely used in the clinical diagnosis of prostatemalignancies.

Other markers for tumors are also known. For example, carcinoembryonicantigen (CEA) is of prognostic value for colorectal carcinoma; in breastcancer, CEA and CA15-3 are used as postoperative markers (Mughal et al.,“Serial Plasma Carcinoembryonic Antigen Measurements During Treatment ofMetastatic Breast Cancer”, JAMA 249:1881-1886 (1983)), but not inpreoperative diagnosis. BRCA1/2 status can be used as a risk factorindicator, HER-2/neu (c-erbB-2) status correlates with relapse andsurvival (Slamon et al., “Human Breast Cancer: Correlation of Relapseand Survival With Amplification of the HER-2/neu Oncogene”, Science235:177-182 (1987)), and Ki-67 is a proliferation marker that is usefulin the determination of the growth fraction of a tumor (Gerdes, “Ki-67and Other Proliferation Markers Useful for Immunohistological Diagnosticand Prognostic Evaluations in Human Malignancies”, Semin. Cancer Biol.1:199-206 (1990)). However, these markers are of limited usefulness intumor detection, diagnosis and prognosis.

BC200 RNA is a 200-nucleotide long, non-translatable RNA that isprevalently expressed in the nervous system of primates, including man.A partial nucleotide sequence of BC200 RNA from monkey brains was firstreported by Watson and Sutcliffe, Molecular & Cellular Biology7,3324-3327 (1987). This 138 nucleotide sequence showed substantialhomology to the Alu left monomer, a sequence that is repeated many timesthroughout the human and other primate genomes. The sequence offull-length BC200 RNA was subsequently reported by Tiedge et al.,“Primary Structure, Neural-Specific Expression, and Dendritic Locationof Human BC200 RNA”, J. Neurosci. 13, 2382-2390 (1993).

The primary sequence of human BC200 RNA is as follows:

[SEQ ID NO 1] XXCCGGGCGCGGUGGCUCACGCCUGUAAUCCCAGCUCUCAGGGAGGCUAAGAGGCGGGAGGAUAGCUUGAGCCCAGGAGUUCGAGACCUGCCUGGGCAAUAUAGCGAGACCCCGUUCUCCAGAAAAAGGAAAAAAAAAACAAAAGACAAAAAAAAAAUAAGCGUAACUUCCCUCAA AGCAACAACCCCCCCCCCCCUUU

Expression of the small neuronal non-coding transcript BC200 RNA, itselfa modulator of translation (Wang, et al., “Dendritic BC1 RNA: FunctionalRole in Regulation of Translation Initiation”, J. Neurosci, vol. 22,pages 10232-10241(2002)), is tightly regulated. The RNA is not normallydetected at higher than background levels in non-neuronal somatic cells(Tiedge et al., supra). However, the tight neuron-specific control ofBC200 RNA expression is deregulated in various tumors, including breasttumors. BC200 RNA is associated with malignancy and is not detectable innormal non-neuronal somatic tissue or in benign tumors such asfibroadenomas of the breast. Lin et al. “Expression of Neural BC200 RNAin Breast Cancer”, Era of Hope Proceedings, Vol. 1, p. 122 (Departmentof Defense, 2000).

U.S. Pat. Nos. 5,670,318 and 5,736,329, the contents of each of whichare incorporated by reference herein, disclose the complete sequence ofhuman BC200 RNA and the use of polynucleotide probes which can be usedto specifically detect the presence of human BC200 RNA in a tissuesample.

The primary sequence of BC200 RNA can be subdivided into threestructural domains. Domain I is nucleotides 1-122 and is substantiallyhomologous to Alu repetitive elements which are found in high copynumbers in primate genomes. However, this region includes two bases notfound in Alu or SRP-RNA, i.e., nucleotides at positions 48 and 49, whichcan be used to develop amplification primers specific to BC200 RNAsequences. Domain II is an A-rich region consisting of nucleotides123-158. Domain III, consisting of nucleotides 159-200, contains aunique sequence with no homology to other known human sequences whichcan be used to identify BC200 RNA in tissues.

Oncological pathologists have long recognized that the differing degreesof malignancy of tumors is reflected in their morphological structure.There are three general grades of tumors, low, intermediate and high,with the high grade typically being associated with the most aggressivetumors (Elston et al., The Breast, Ch. 17, pp. 365-383, (1998)).

Ductal carcinoma in situ (DCIS) is a common but heterogeneous group ofneoplastic diseases. Approximately 25% of DCIS will develop intoinvasive carcinomas within 15 years if left untreated (Elston et al.,The Breast, Ch. 14, pp. 249-281, (1998)). However, to date there is noreliable indicator, molecular or otherwise, to predict the invasivepotential of a given DCIS. Most women therefore elect to have DCISremoved surgically, which in many instances is unnecessary and amountsto over-treatment (Ernster, “Increases in Ductal Carcinoma In Situ inRelation to Mammography: A Dilemma”, NIH Consensus DevelopmentConference on Breast Cancer Screening for Women Ages 40-49, pp. 147-151(NIH, 1997)).

Clearly, a reliable prognostic indicator of cancer, including DCIS,would be most valuable in assisting physicians and patients in makinginformed treatment decisions.

SUMMARY OF THE INVENTION

The present invention is directed to methods for diagnosing invasivecarcinomas and determining the likelihood that a carcinoma which is notyet invasive is likely to become an invasive carcinoma. The presentinvention is also directed to methods for determining the tumor grade ofa sample.

The methods include: obtaining a physiological sample from a human;preparing the test sample such that RNA in the test sample is capable ofreacting with a detection reagent possessing a labeling signal;combining the test sample with the detection reagent under conditionsthat produce a detectable reaction product if human BC200 RNA ispresent; measuring the amount of detectable reaction product by itslabeling signal; comparing the level of labeling signal to a labelingsignal from a non-high grade carcinoma control or any other suitablecontrol such as normal tissue that does not express BC200 RNA; andcorrelating an elevated labeling signal in the test sample with adetermination that the carcinoma is of a grade likely to becomeinvasive.

Where the method is utilized for diagnosing an invasive carcinoma, thelabeling signal from a test sample is compared to a labeling signal froma non-invasive carcinoma control. An elevated signal in the test samplecompared to a non-invasive carcinoma control indicates the presence ofan invasive carcinoma in the sample.

Where the method is directed to determining tumor grade, the labelingsignal in the test sample is compared correlated with the labelingsignal in a non-high grade carcinoma control and/or anintermediate-grade carcinoma control prepared using the same steps asthe test sample. A relative same level of labeling signal in the testsample and a low-grade carcinoma control indicates a low-grade carcinomain the test sample. Where an elevated amount of labeling signal in thetest sample is present compared to the amount of labeling signal from alow-grade carcinoma control, at least an intermediate-grade carcinoma inthe test sample is indicated. In such a case, the test sample may becompared with an intermediate-grade carcinoma control prepared using thesame steps as the test sample. Where an elevated amount of labelingsignal in the test sample is present compared to an intermediate-gradecarcinoma control, a high grade carcinoma in the test sample isindicated.

In one embodiment, the detection reagent utilized in the methods of thepresent invention is an oligonucleotide probe capable of hybridizingwith human BC200 RNA and RT-PCR is utilized to produce a detectablereaction product if human BC200 RNA is present.

Preferably, the amount of labeling signal is measured by a techniqueselected from the group consisting of emulsion autoradiography,phosphorimaging, light microscopy, confocal microscopy, multi-photonmicroscopy, and fluorescence microscopy. Quantitative analysis may beconducted to determine the labeling signal intensity, which may then beutilized in the diagnosis and prognosis of carcinomas.

Preferably, carcinomas and/or tumors diagnosed in accordance with themethods of the present invention may be carcinomas of the breastincluding, but not limited to, carcinomas in situ, which in turn includeductal carcinoma in situ and lobular carcinoma in situ; infiltratingcarcinomas, which in turn include infiltrating ductal carcinomas andinfiltrating lobular carcinomas such as tubulo-lobular carcinomas;mucinous carcinomas; and medullary carcinomas. Other carcinomasmonitored in accordance with the methods of the present inventioninclude, but are not limited to, tumors of the skin, kidney, parotidgland, lung, bladder and prostate.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been surprisingly foundthat not only may BC200 RNA be used to detect various carcinomas but, inaddition, by monitoring BC200 RNA levels, tumor grade may be determinedas well as the likelihood that a carcinoma is of a grade likely tobecome invasive.

Carcinomas which may be monitored and diagnosed in accordance with thepresent invention are varied and include, but are not limited to,carcinomas of the breast, such as infiltrating mammary carcinomas, e.g.,infiltrating ductal carcinomas (IDC) and infiltrating lobular carcinomas(ILC), which in turn include tubulo-lobular carcinomas; carcinomas insitu, e.g., ductal carcinoma in situ (DCIS) and lobular carcinomas insitu (LCIS); mucinous carcinomas; medullary carcinomas; and others.Other carcinomas which may be monitored and diagnosed include, but arenot limited to, carcinomas of the skin, kidney, parotid gland, lung,bladder and prostate.

It should be noted that BC200 RNA can not be classified as aproliferation marker, for two reasons. First, of all normal somaticcells, only neurons express BC200 RNA. Most neurons are post-mitotic anddo not proliferate. Secondly, proliferating somatic cells, with theexception of cancer cells such as mammary carcinoma cells, do notexpress BC200 RNA. Thus, BC200 expression is not associated withproliferation per se, but rather with specific malignancies, inparticular with invasive potential.

In one embodiment, the present invention provides a prognostic methodfor determining the likelihood that a carcinoma is of a grade likely tobecome an invasive carcinoma. This method may also be used in thediagnosis of invasive tumors. This method involves obtaining aphysiological test sample from a human, preparing the test sample suchthat RNA in the test sample is capable of reacting with a detectionreagent possessing a labeling signal, combining the test sample with thedetection reagent under conditions that produce a detectable reactionproduct if human BC200 RNA is present, measuring the amount ofdetectable reaction product by its labeling signal, comparing the amountof labeling signal for the test sample to a labeling signal from anon-high grade carcinoma control prepared using the same steps as thetest sample, and correlating an elevated amount of labeling signal inthe test sample with a determination that the carcinoma is likely tobecome invasive.

In another embodiment, the present invention provides a method fordetermining tumor grade. This method involves obtaining a physiologicaltest sample from a human, preparing the test sample such that RNA in thetest sample is capable of reacting with a detection reagent possessing alabeling signal, combining the test sample with the detection reagentunder conditions that produce a detectable reaction product if humanBC200 RNA is present, measuring the amount of detectable reactionproduct by its labeling signal, and comparing the amount of labelingsignal measured in the test sample to a labeling signal from a non-highgrade carcinoma control prepared using the same steps as the testsample. Relatively the same level of labeling signal in the test sampleand a low-grade carcinoma control prepared using the same steps as thetest sample indicates a low-grade carcinoma in the test sample; anelevated amount of labeling signal in the test sample compared to theamount of labeling signal from a low-grade carcinoma control preparedusing the same steps as the test sample indicates at least anintermediate-grade carcinoma in the test sample; and an elevated amountof labeling signal in the test sample compared to an intermediate-gradecarcinoma control prepared using the same steps as the test sampleindicates a high grade carcinoma in the test sample.

Preferably, the detection reagent utilized in the methods of the presentinvention is an oligonucleotide probe capable of hybridizing with humanBC200 RNA, and RT-PCR is utilized to produce a detectable reactionproduct if human BC200 RNA is present in a test sample. Most preferably,the detectable reaction product possesses a labeling signal, such as afluorescent signal.

Examples of oligonucleotide probes which may be used to monitor thelevel of BC200 RNA in a sample are described in U.S. Pat. Nos. 5,670,318and 5,736,329. Such probes are complementary to the unique sequences ofDomain III of human BC200 RNA, or to its corresponding chromosomal DNA,i.e., complementary to at least a portion of the sequence:

UAAGCGUAAC UUCCCUCAAA GCAACAACCC [SEQ ID NO 2] CCCCCCCCCU UU 42The probes may be linear oligonucleotides containing from about 10 to 60bases. The length must be sufficient to provide a reasonable degree ofspecificity such that binding with BC200 RNA will be preferred overbinding to other polynucleotides.

As used herein, the term “oligonucleotide probe” refers to either a DNAor an RNA probe.

One probe within the scope of the invention is complementary to thenucleotides 156-185 of BC200 RNA. This 30-nucleotide probe has thesequence:

TTGTTGCTTT GAGGGAAGTT ACGCTTATTT 30 [SEQ ID NO 3]Another useful probe is a 21-nucleotide probe complementary tonucleotides 158-178, i.e.:

TTTGAGGGAA GTTACGCTTA T 21 [SEQ ID NO 4]As is apparent, suitable probes may be complementary with the portionsof BC200 RNA outside Domain III, provided they are also complementary toa portion (i.e., at least about 10 bases) of the unique Domain IIIsufficient to provide specificity. Probes may also be complementary toportions of Domain III alone. A second class of probes may also be usedwhich are complementary to a portion of Domain II, spanning nucleotides146-148. The above probes may be used for detection of BC200 RNA or asamplification primers.

In another aspect of the invention, probes can be utilized which arecomplementary to, and specifically hybridize with, a portion of theAlu-repetitive sequence spanning the two unique nucleotides at positions48 and 49 of BC200 RNA or its corresponding DNA. Examples of such probesare:

CCTCTTAGCC TCCCTGAGAG CT 22 [SEQ ID NO 5]an antisense probe that will bind BC200 RNA and:

CCAGCTCTCA GGGAGGCTAA 20 [SEQ ID NO 6]a sense probe that will bind to corresponding DNA sequences. Theseprobes can be used for detection or as amplification primers.

The probes utilized in accordance with the present invention can be madeby any of a variety of techniques known in the art. For example, RNAprobes can be generated by in vitro transcription. In this approach, thedesired sequence is first cloned into a suitable transcription vector(e.g., pBluescript). This vector is linearized so that transcriptionwill terminate at a specific location, and RNA is transcribed from suchlinearized templates, using SP6, T3, or T7 RNA polymerase. The probescan be ³⁵S- or ³H-labeled by adding the appropriate radiolabeledprecursors to the reaction mixture. Template DNA is then digested withDNase I. RNA probes can be further purified by gel filtration or gelelectrophoresis.

Probes can also be made by oligolabeling, although this technique ismore suited to longer nucleic acid polymers. In this method, doublestranded DNA is first denatured. Random sequence oligonucleotides arethen used as primers for the template directed synthesis of DNA. TheKlenow fragment of E. coli DNA polymerase I is frequently used in thisapplication. Reverse transcriptase can be used if the template is RNA.Labeling of the probe is achieved by incorporation of radiolabelednucleotides, e.g., [α-³²P]dNTps.

Another approach for generation of probes is nick translation. Doublestranded DNA is used in this method. Nicks (gaps in one strand) areintroduced by DNase I. E. coli DNA polymerase I is used simultaneouslyto add nucleotide residues to the 3′ termini of the DNA at the nickpoints. Incorporation of radiolabeled precursor nucleotides results inthe uniform labeling of the probe. Probes contain both strands.

Single stranded DNA probes can be made from templates derived frombacteriophage M13 or similar vectors. An oligonucleotide primer,complementary to a specific segment of the template, is then used withthe Klenow fragment of E. coli DNA polymerase I to generate aradiolabeled strand complementary to the template. The probe is purifiedfor example by gel electrophoresis under denaturing conditions.

Oligonucleotides of any desired sequence can also be synthesizedchemically. Solid phase methods are routinely used in the automatedsynthesis of oligonucleotides.

Probes useful in accordance with the present disclosure can be labeled.A variety of enzymes can be used to attach radiolabels (using dNTPprecursors) to DNA termini. The 3′ termini of double stranded DNA canfor example be labeled by using the Klenow fragment of E. coli DNApolymerase I. Blunt ended DNA or recessed 3′ termini are appropriatesubstrates. T4 DNA polymerase can also be used to label protruding 3′ends. T4 polynucleotide kinase can be used to transfer a ³²P-phosphategroup to the 5′ termini of DNA. This reaction is particularly useful tolabel single stranded oligonucleotides. Probes can also be labeled viaPCR labeling in which labeled nucleic acids and/or labeled primers areused in PCR generation of probes from an appropriate clone. See Kelly etal., Genomics 13: 381-388 (1992).

The methods of the present invention utilize oligonucleotide probes likethose described above to screen tissue for the presence of BC 200 RNAwhich, itself, is utilized to diagnose carcinomas and determine whetheror not it is likely that a given carcinoma in situ will progress to aninvasive carcinoma.

The basic methodology of the screening procedure involves the followingsteps:

-   (1) obtaining a physiological sample;-   (2) treating the sample to render RNA and/or DNA available for    hybridization;-   (3) hybridizing the treated sample with a probe specific for Domain    III of human BC200 RNA; and-   (4) analyzing for the occurrence of hybridization.

Suitable physiological samples include biopsy specimens, such assurgical specimens, blood, and other body fluids including, but notlimited to, nipple discharges, sputum, semen, and scrapings.

While the method employed to treat the tissue sample is not critical,provided that nucleic acids in the sample are made available forhybridization, several specific options are worth noting. Directisolation of RNA by the guanidine thiocyanate method followed byCsCl-density gradient centrifugation may be effective in many cases,particularly for isolation of RNA from biopsy specimens. Where thesample size is small, however, amplification of the RNA may bedesirable.

Amplification of the RNA can be achieved by first lysing cells in thesample to render RNA available for hybridization. This can beaccomplished by (1) extraction of RNA with guanidinium thiocyanate,followed by centrifugation in cesium chloride; (2) extraction of RNAwith guanidine HCl and organic solvents; or (3) extraction of RNA withmild detergents (such as NP-40), combined with proteinase digestion.These and other RNA extraction methods are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual, 3d ed., Cold Spring HarborLaboratory Press (2001). The isolated RNA is converted into cDNA whichis then amplified using probes selective for the 3′ end of BC200 RNAsequence. (See U.S. Pat. No. 4,683,202, the contents of which areincorporated by reference herein.) cDNA may also be amplified usingligase-based methods (Barany et al., “Genetic Disease Detection and DNAAmplification Using Cloned Thermostable Ligase”, Proc. Nat'l. Acad. Sci.USA 88, 189-193 (1991)) or isothermal transcription-based methods (Kwohet al., “Transcription-based Amplification System and Detection ofAmplified Human Immunodeficiency Virus Type 1 With a Bead-based SandwichHybridization Format”, Proc. Nat'l. Acad. Sci. USA 86, 1173-1177(1989)). The amplified DNA can then be detected directly via anappropriate probe.

The hybridization can be carried out using any of the numerousprocedures known for assaying for nucleic acids. These include variousblot techniques (i.e., dot, Northern, Southern, etc.), and sandwichbased techniques such as those described in U.S. Pat. Nos. 4,486,539;4,751,177; 4,868,105; 4,894,325 and European Patent Publication 0 238332 (the contents of each of which are incorporated by referenceherein). To facilitate detection, the probe may have a label, such as aradiolabel, chemiluminescent label, fluorescent label or chromogeniclabel, or an immobilization moiety. Probes modified with biotin ordigoxygenin, which can serve as either a detectable label or animmobilization moiety, may be particularly useful.

In addition, other techniques such as reverse transcription polymerasechain reaction (RT-PCR) may be utilized to detect BC200 RNA in smallsamples, such as those obtained by fine needle or core needle biopsies,or those obtained from body fluids such as blood or nipple discharges.

In conventional PCR assays, oligonucleotide primers are designedcomplementary to the 5′ and 3′ ends of a DNA or RNA sequence ofinterest. During thermal cycling, DNA or RNA is heat denatured. Thesample is then brought to annealing and extension temperatures in whichthe primers bind their specific complements and are extended by theaddition of nucleotide tri-phosphates by a polymerase, such as Taqpolymerase. With repeated thermal cycling, the amount of template DNA orRNA is amplified.

In RT-PCR, such as those using TaqMan® chemistry, an oligonucleotideprobe may be designed that is complementary to the sequence regionbetween the primers within the PCR amplicon. The probe may contain afluorescent reporter dye at its 5′ end and a quencher dye at its 3′ end.When the probe is intact, its fluorescent emissions are quenched by thephenomena of fluorescent resonance energy transfer (FRET). Duringthermal cycling, the probe hybridizes to the target DNA or RNAdownstream of one of the primers. TaqMan® chemistry relies on the 5′exonuclease activity of Taq polymerase to cleave the fluorescent dyefrom the probe. As PCR product accumulates, fluorescent signal isincreased. By measuring this signal, the amplified product can thus bequantified which allows for the quantitation of RNA present in a sample.In combination with the PCR primers, the probe provides another level ofspecificity in assays to differentiate and quantify the BC200 RNA.

Different primer pairs may be utilized to amplify BC200 RNA by RT-PCR.The 5′ portion of BC200 RNA is similar in sequence to human Alu-Jrepetitive elements. However, BC200 RNA differs from these elements byfour nucleotide differences and two tandem base insertions betweenpositions 35-50. Therefore, in one embodiment 5′ primers are designed totarget this region. The sixty 3′-most nucleotides (nucleotides 141-200)of BC200 RNA are unique, and have been used successfully as a specificprobe in Northern and Southern analyses (Tiedge et al., “PrimaryStructure, Neural-Specific Expression, and Dendritic Location of HumanBC200 RNA”, J. Neurosci. 13, 2382-2390 (1993)).

Preferably, RT-PCR is conducted to detect BC200 RNA, and its fluorescentsignal is utilized to determine the invasive potential of a carcinoma.

Signal intensities may be determined by methods known to those skilledin the art including, but not limited to, emulsion autoradiography,phosphorimaging, light microscopy, confocal microscopy, (e.g. confocallaser scanning microscopy) and multi-photon microscopy. In the case ofnon-radioactive labeling techniques (e.g., using biotin or digoxygenin),light microscopy or fluorescence microscopy may be used.

The labeling intensity for a given sample may then be compared with alabeling signal from a non-high grade carcinoma control. An elevatedlabeling signal for a test sample compared to the signal from a non-highgrade carcinoma control will result in a determination that a givensample contains a carcinoma that is likely to become invasive.Conversely, a low or equivalent labeling signal compared to the signalfrom a non-high grade carcinoma control will result in a determinationthat a given sample is either free of carcinoma or, where the source ofthe sample is a tumor, possesses a carcinoma that is unlikely to becomeinvasive.

Similarly, the labeling signal of a test sample may be compared to alabeling signal from a non-invasive carcinoma control prepared using thesame steps as the test sample. An elevated signal in a test samplecompared to the labeling signal from a non-invasive carcinoma controlmay be used to diagnose an invasive carcinoma. Conversely, a low orequivalent signal compared to the non-invasive carcinoma control willresult in a determination that the sample does not contain an invasivecarcinoma.

In one embodiment, where autoradiography is used to determine the signalintensity in a test sample, the autoradiographs may be subjected toquantitative analysis, preferably using commercially or publiclyavailable image analysis software such as MetaMorph Software (UniversalImaging Corp., Downingtown, Pa.), or NIH Image Software (NIH, Bethesda,Md.). Confocal and multi-photon microscopy may also be used forquantitative analysis. Hand counting may also be used for quantitativeanalysis, and was the traditional method prior to the introduction ofimage analysis software.

The resulting labeling signal or signal intensity of a sample may beexpressed as “autoradiographic labeling units” or “ALUs”, which arerelative units equivalent to the number of silver grains per unit areain the autoradiograph which, in turn, is equivalent to the amount oflabeling signal. In one embodiment, where autoradiography is used inconjunction with image analysis software (preferably MetaMorph Software)in the quantitative analysis of the signal intensity of a sample, a testsample possessing a labeling signal of greater than about 1000 ALUs maybe used to diagnose a high grade or invasive carcinoma. If a carcinomais not already invasive, there is a high probability a test samplepossessing a labeling signal of greater than about 1000 ALUs contains acarcinoma that will progress to an invasive carcinoma. Conversely, alabeling signal of less than about 1000 ALUs may be used to diagnose anon-invasive carcinoma or a non-high grade carcinoma possessing a lowprobability the carcinoma will progress to an invasive carcinoma. AsALUs are relative units, a different sampling area or the use ofdifferent image analysis software may provide a different number.However, as one skilled in the art would readily recognize, relativeunits may be utilized to compare a test sample with a control anddetermine whether or not a sample does, in fact, possess an invasivecarcinoma or a carcinoma that is likely to progress to an invasivecarcinoma.

Similarly, in another embodiment, the present invention is directed tomethods for grading tumors. The labeling intensity for a given tumorsample may be compared with a labeling signal in a low-grade carcinomacontrol. Where the level of labeling signal in a test sample isrelatively the same as the labeling signal of a low-grade tumor control,a low-grade tumor is indicated. However, where an elevated amount oflabeling signal in a test sample is present compared to the amount oflabeling signal from a low-grade tumor control, at least anintermediate-grade tumor in the test sample is indicated. In such acase, a test sample may then be compared with an intermediate-gradetumor control prepared using the same steps as the test sample. Where anelevated amount of labeling signal in the test sample is presentcompared to an intermediate-grade tumor control, a high grade tumor inthe test sample is indicated. By determining the amount of BC200 RNApresent in a tumor, the tumor may be graded and a determination made asto whether or not it is likely the tumor will become invasive.

In one preferred embodiment, the methods of the present invention areused in the diagnosis and prognosis of DCIS. In DCIS, BC200 RNAexpression is dependent on tumor grade. Comparative genomichybridization has shown that the transition from DCIS to invasivecarcinoma follows a specific genetic pathway that appears to beassociated with differentiation status and grade (Buerger et al.,“Comparative Genomic Hybridization of Ductal Carcinoma In Situ of theBreast—Evidence of Multiple Genetic Pathways”, 187 J. Pathol. 396-402(1999); Buerger et al., “Different Genetic Pathways in the Evolution ofInvasive Breast Cancer are Associated With Distinct MorphologicalSubtypes”, 189 J. Pathol. 521-526 (1999)).

The probes of the invention may be supplied as part of a kit forscreening tissue, such as breast tissue, for BC200 RNA. In addition tothe probe or other detection reagent that produces a diagnostic reactionproduct if BC200 RNA is present, such a kit may include one or more ofthe following:

-   (1) a solid support to which the diagnostic reaction product nucleic    acids are affixed during the screening procedure;-   (2) amplification primers and enzymes for amplification of nucleic    acids in a sample;-   (3) a labeled reagent that reacts with the diagnostic reaction    product to render it detectable; and-   (4) solutions effective to lyse the physiological sample to render    RNA available for hybridization.

Suitable amplification primers include those identified in the Examples,as well as others which will result in amplification, if present, ofDomain III of BC200 RNA, possibly together with portions of Domains IIand I. A particularly preferred 5′-amplification primer is one that iscomplementary to a portion of Domain I of BC200 RNA, or thecorresponding cDNA, that includes the unique nucleotides at positions 48and 49. Suitable enzymes include reverse transcriptase, Taq polymerase,rTth DNA polymerase and RNA polymerase.

In accordance with the present invention, it has been surprisinglydiscovered that the amounts of BC200 RNA expressed by cancerous tumorcells of the breast correlate with tumor type, grade and stage. Thus,BC200 RNA is expressed at high levels in invasive carcinomas.Accordingly, BC200 RNA is used in accordance with the present inventionas a molecular indicator in the diagnosis and prognosis of invasivecarcinomas, including carcinomas of the breast. The correlation betweenBC200 RNA expression levels and tumor grade can be used as a molecularindicator of invasive potential. High BC200 RNA levels in a carcinomaindicate a high likelihood of a future invasive carcinoma in thatpatient. BC200 RNA expression is therefore a valuable tool to predicttumor progression.

It will be understood that various modifications may be made to theembodiments described herein. Therefore, the above description shouldnot be construed as limiting, but merely as exemplifications ofpreferred embodiments. For example, while the invention is describedprincipally in terms of using oligonucleotide hybridization probes orRT-PCR to detect BC200 RNA levels in those suffering from carcinoma,such as carcinoma of the breast, it will be appreciated that thebeneficial result of screening for neoplastic diseases can be achievedusing any detection technique. For example, RNA-specific antibodies toBC200 RNA could be used, e.g., in an ELISA assay, to detect BC200 RNA intissue samples. See Uchiumi et al., “A Human Autoantibody Specific for aUnique Conserved Region of 28 S Ribosomal RNA Inhibits the Interactionof Elongation Factors 1 alpha and 2 With Ribosomes”, J. Biol. Chem. 266:2054-62 (1991). Peptide nucleic acids that hybridize with BC200 RNA mayalso be used as diagnostic reagents. See Hanvey et al., “Antisense andAntigene Properties of Peptide Nucleic Acids”, Science 258: 1481-1485(1992). Similarly, BC200 RNA may be complexed with proteins in vivo toform a ribonucleoprotein (“RNP”). Antibodies specific to BC200 RNA couldthen be used in an immunoassay detection scheme. Those skilled in theart will envision other modifications within the scope and spirit of thefeatures described herein.

EXAMPLE 1 Amplification of BC200 RNA

The 5′ and 3′ Domains of BC200 RNA were amplified separately. Foramplification of the 5′ BC200 RNA sequence, 1 μg total RNA was isolatedfrom human neocortex using the guanidinium thiocyanate method followedby phenol extraction and CsCl centrifugation, and converted into firststrand cDNA using the thermostable rTth DNA polymerase (Perkin ElmerCetus) according to the instructions of the manufacturer. The primerused in this step was:

GTTGTTGCTT TGAGGGAAG 19 [SEQ ID NO 7]

The 3′ end of the product was then T-tailed using dTTP and terminaltransferase (Boehringer Mannheim). The tailed cDNA was PCR-amplified(Frohman et al., Proc. Nat'l. Acad. Sci. USA 85: 8998-9002 (1988)) in 30cycles (denaturation for 30 seconds at 94° C., annealing for 1 minute at55° C., extension for 2 minutes at 72° C.; initial denaturation was for4 minutes at 94° C., final extension was for 10 minutes at 72° C.),using the primers:

GCCTTCGAAT TCAGCACCGA GGGAAGTTAC [SEQ ID NO 8] GCTTA 35 and GCCTTCGAATCAGCACCAA AAAAAAAAAA [SEQ ID NO 9] AAAAA 35The products were further amplified in a second set of 30 cycles(conditions see above), using the adapter primer:

GCCTTCGAAT TCAGCACC 18 [SEQ ID NO 10]After digestion with EcoRI, the PCR-products were cloned into the EcoRIsite of λ ZAPII (Stratagene) following the manual of the manufacturer.10³ plaques were screened with an internal oligonucleotide probe:

AAAAAAAAA(T/A) (T/G)GCCGGGCGC GGT 23 [SEQ ID NO 11]and 6 positive clones were sequenced.

For amplification of the 3′ BC200 RNA sequence, 10 μg total RNA fromhuman neocortex were A-tailed using poly A polymerase (DeChiara et al.,“Neural BC1 RNA: cDNA Clones Reveal Nonrepetitive Sequence Content”,Proc. Natl. Acad. Sci. USA 84, 2624-2628 (1987)). Tailed RNA was thenconverted into first strand cDNA with reverse transcriptase in thepresence of MeHgOH (Invitrogen), using the primer:

GCCTTCGAAT TCAGCACCTT TTTTTTTTTT [SEQ ID NO 12] TTTTT 35This primer, in combination with the primer:

GCCTTCGAAT TCAGCACCAA AATAAGCGTA [SEQ ID NO 13] ACTTCCC 37was also used for PCR-amplification (see above). Products were clonedinto λ ZAPII (see above), and 14 clones that were identified with SEQ IDNO 13 were sequenced using the enzymatic chain termination reaction.

EXAMPLE 2 Production of BC200 RNA Specific Probe

Two types of probes have routinely been used. An oligodeoxynucleotide ofthe desired sequence was chemically synthesized and purified bychromatography or gel electrophoresis. The oligonucleotide was thenradiolabeled by phosphorylation of the 5′ end. This was achieved byusing the enzyme polynucleotide kinase with γ³²P-labeled ATP. Theradiolabeled probe (specific activity: >10⁸ cpm/μg) was separated fromunincorporated label by gel filtration, and the probe was used at aconcentration of 10⁶ cpm/ml.

In addition, RNA probes were generated by in vitro transcription. Inthis approach, the desired sequence was first cloned into a suitabletranscription vector (e.g., pBluescript). This vector was thenlinearized (so that transcription would terminate at a desiredlocation), and RNA was transcribed from such linearized templates, usingSP6, T3, or T7 RNA polymerase. ³⁵S- or ³H-UTP was present during thetranscription reaction, and the resulting probes were thus ³⁵S- or³H-labeled. Template DNA was then digested with DNase I, proteins werephenol-extracted, and the probes were ethanol-precipitated. RNA probeswere used for in situ hybridization experiments.

EXAMPLE 3

To capture BC200 RNA from blood (Gillespie et al., “Dissolve andCapture: A Strategy for Analyzing mRNA in Blood”, Nature 367, 390-391(1994)), biotinylated 2′ O-alkyl oligoribonucleotides (Iribarren et al.,“2′-O-Alkyl Oligoribonucleotides as Antisense Probes”, Proc. Natl. Acad.Sci. USA 87, 7747-7751 (1990); Lamond et al., “AntisenseOligoribonucleotides Made of 2′-O Alkyl RNA: Their Properties andApplications in RNA Biochemistry”, FEBS Lett. 325, 123-127 (1993))previously used for the concentration and purification of BC1 RNA andBC200 RNA are used. For the analysis of BC200 RNA in blood from breastcancer patients, RNA is captured onto antisense oligoribonucleotidescoupled to a matrix through biotin-streptavidin binding. Followingrepeated washes, captured BC200 RNA is eluted and amplified by RT-PCR.

PCR cycling parameters such as range of annealing temperatures andnumber of cycles are determined (12-40 cycles, exponential phase andplateau). This is followed by electrophoresis of PCR samples, filtertransfer, and hybridization with probes for BC200 RNA and cyclophilinmRNA.

The most efficient PCR primers are selected and optimal PCR conditionsare established to determine the ideal conditions for processing RNAthat is obtained from fine needle aspirants and core biopsies. Routineshort or one-step protocols are used that will most effectively rid thesample of genomic DNA. To ascertain the value of the optimized RT-PCRprocedure, samples of human core biopsy material are tested (1-1.5 mm indiameter, 1.7-1.9 cm length). This amount of tissue is sufficient toanalyze the RNA content by both PCR and in situ hybridization, inparallel with conventional histopathological methods.

Because BC200 RNA is short and intronless, a negative control withoutreverse transcriptase (RT) accompanies every experimental sample. Asecond, positive control is needed to detect false negatives, in case ofdegraded RNA or of suboptimal RT reaction. Cyclophilin mRNA is chosen tobe the reporter RNA used as an internal gauge for efficiency of firststrand synthesis. Previous results show that the cyclophilin mRNA isexpressed at the same unvarying levels in normal and tumor tissues alike(Chen et al., “Expression of Neural BC200 RNA in Human Tumors”, J.Pathol. 183, 345-351 (1997)). The purpose of this control experiment isto monitor the overall efficiency of the entire procedure, as performedon the individual sample.

Cyclophilin mRNA primers are tested not only for efficiency ofamplification, but also for lack of interference with the ongoing BC200RNA amplification. The primer pairs for BC200 RNA and for cyclophilinmRNA are selected to amplify with similar efficiency, in the same or inseparate tubes. To test the efficiency of primer pairs, RNA samples areused from normal and tumor tissues that are previously tested byNorthern or in situ hybridization, and where the relative signal ofBC200 RNA (or cyclophilin mRNA) from sample to sample is known, assuggested by Chen et al., supra.

EXAMPLE 4

Human breast tissue was prepared for evaluation. Tissue was obtained andtreated to render RNA and/or DNA available for hybridization by freezingin liquid nitrogen. Samples were cryo-embedded in Tissue-Tek OCTembedding medium (Miles, Elkhart, Ind.), frozen in liquid nitrogen, andstored at −80° C. before being sectioned in a Bright Microtome Cryostatat 10 μm thickness following the procedures set forth in Tiedge, H.,“The Use of UV Light as a Cross-linking Agent for Cells and TissueSections In in situ Hybridization”, DNA Cell Biol. 10, 143-147 (1991).Sections were thaw-mounted onto gelatin/poly-L-lysine coated microscopeslides and stored at −80° C. until further processing. The samples werethen subjected to in situ hybridization with a ³⁵S-labeled RNA probespecific for Domain III of human BC200 RNA human RNA produced inaccordance with Example 2 above. In situ hybridization was performed asdescribed in Tiedge et al., “Primary Structure, Neural-SpecificExpression, and Dendritic Location of Human BC200 RNA”, J. Neurosci. 13,2382-2390 (1993). The final high-stringency wash was performed in0.1×SSC, 0.05% sodium pyrophosphate, 14 mM 2-mercaptoethanol at 37° C.as described in Tiedge, H., “The Use of UV Light as a Cross-linkingAgent for Cells and Tissue Sections In in situ Hybridization”, DNA CellBiol. 10, 143-147 (1991).

Tables 1 and 2 below provide semi-quantitative evaluations of the BC200RNA signals obtained from these samples of breast tissue. The symbols+/−, +, ++, +++, ++++ reflect increasing levels of RNA detected. Thediagnosis, grading and classification of the tissue were independentlyestablished by cancer pathologists.

TABLE 1 Case Number Diagnosis Grading Signal 1 Fibroadenoma − 2Fibroadenoma − 3 Fibroadenoma − 4 Fibroadenoma − 5 DCIS Non-high grade+/− 6 DCIS Non-high grade +/− 7 DCIS High grade + 8 DCIS High grade + 9DCIS High grade + 10 DCIS High grade ++ 11 DCIS High grade ++

TABLE 2 Case Number Diagnosis TNM Classification Signal 12 Invasiveductal carcinoma T1c, Nx, Mx, HD 3 ++ (with ductal hyperplasia) 13Mixed-differentiated T1b, Nx, Mx, HD 2 ++ carcinoma 14 Invasive ductalcarcinoma T1c, Nx, Mx, HD 3 ++ 15 DCIS (Comedo type) Tis, N0, Mx, HD 3++ 16 Invasive lobular carcinoma T1c, Nx, Mx, HD 2 +++ 17 Invasivelobular carcinoma T1c, Nx, Mx, HD 2 +++ (with papilloma) 18 Invasiveductal carcinoma T1c, Nx, Mx, HD 2 +++ (with DCIS) 19 Invasive lobularcarcinoma T1c, Nx, Mx, HD 2 +++ (with LCIS) 20 Invasive ductal carcinomaT2, Nx, Mx, HD 2 +++ (with DCIS) 21 Invasive lobular carcinoma T3, Nx,Mx, HD 3 +++ (with DCIS) 22 Invasive ductal carcinoma T2, Nx, Mx, HD 2+++ (with DCIS, Comedo type) 23 Invasive lobular carcinoma T1c, Nx, Mx,+++ (with fibrocystic mastopathy) 24 Invasive ductal carcinoma T4a, Nx,M1, HD 3 ++++ 25 Tubulobular carcinoma T4d, Nx, Mx, HD 2 ++++ (withDCIS)TNM Classification is the most widely used means for classifying theextent of cancer spread. TNM classification is based on tumor size,number of involved lymph nodes, and number of distant metastases, alsoknown as the tumor-nodes-metastasis (TNM) system. (X means notestablished.) This system has been adopted by the International Unionagainst Cancer and the American Joint Committee on Cancer as originallypublished in the 1992 Manual for Staging of Cancer, 4^(th) ed. (Beahrs,O. H., et al.), pp. 149-154 (Philadelphia, Lippincott 1992).

Signal intensities for the above samples were determined by emulsionautoradiography. In particular, signal intensities were obtained for 3non-high grade DCIS, 4 high-grade DCIS, 5 normal (i.e. non-tumor) tissuesamples, and 5 fibroadenomas. Quantitative analysis of autoradiographicsilver grain density was performed using MetaMorph software (UniversalImaging Corp., Downingtown, Pa.). Quantitative imaging results weregiven in the format mean±standard error mean (s.e.m.) in relative units(ALUs). The average labeling intensity determined for non-high gradeDCIS was 408±150 ALUs whereas for high grade DCIS, it was 3262±923 ALUs.Normal (i.e. non-tumor) tissue was scored at 206±57 ALUs. Fibroadenomaspossessed an average labeling intensity of 502±120 ALUs. Statisticalanalysis showed that the high grade DCIS signal differed significantly(P<0.05) from non-high grade DCIS signal as well as from normal tissuesignal. The non-high grade DCIS signal did not differ significantly fromnormal tissue signal (P=0.31). Therefore, the labeling intensity ofhybridized BC200 RNA provides a categorical (rather than incremental)marker in determining whether or not a DCIS is of a grade likely tobecome invasive.

In invasive ductal carcinomas (IDC) (ductal carcinoma NST) andinfiltrating lobular carcinomas (ILC) including tubulo-lobularcarcinomas, high levels of BC200 RNA expression were also found.Invasive carcinomas had high relative signal intensities of 1800-4000ALUs, depending on tumor type, grade and stage. These values weresignificantly different (P<0.05) from normal tissue signal andfibroadenoma signal. Thus, non-high grade invasive ductal carcinoma wasscored at 1823±478 ALUs (8 cases), high-grade invasive ductal carcinomaat 2957±810 ALUs (8 cases). Both values were significantly different(P<0.05) from normal tissue signal and fibroadenoma signal.

Based upon the above, in accordance with the present invention alabeling signal of greater than about 1000 ALUs determined byautoradiography as described above may be used to predict a high gradeDCIS, which has a high probability that it will progress to an invasivecarcinoma. A signal of less than about 1000 ALUs may be used to predicta non-high grade DCIS, which has a low probability that it will progressto an invasive carcinoma. A signal intensity of greater than about 1000ALUs may also be utilized to diagnose invasive carcinoma such as IDC andILC.

The foregoing results indicate that BC200 RNA levels may be utilized notonly in the diagnosis of invasive carcinoma but also for determiningwhether or not it is likely a carcinoma will progress to an invasivecarcinoma. Moreover, it has been discovered that labeling signals forhigh grade DCIS are significantly higher than in non-high grade DCIS,and approach labeling intensities typical for, and in fact notdistinguishable from, invasive carcinomas.

Therefore, BC200 RNA can also be used as an indicator of tumor grade(low grade vs. intermediate grade vs. high grade) and may be utilized inboth the diagnostis of invasive carcinomas and the prognosis ofcarcinomas not yet invasive.

1. A method for determining whether a breast carcinoma has an invasivephenotype comprising: a) obtaining a physiological test samplecomprising breast carcinoma cells from a human; b) preparing the testsample such that RNA in the test sample is capable of binding with adetection reagent possessing a labeling signal; c) combining the testsample with the detection reagent under conditions that produce adetectable reaction product if human BC200 RNA is present; d) measuringthe amount of detectable labeling signal associated with the testsample; and e) comparing the amount of labeling signal measured in d) toa labeling signal from a non-invasive non-high grade breast tumorcontrol having less than about 1000 ALU units as measured using an imageanalysis software prepared using the same steps as the test sample;wherein a test sample having the same or lower level of labeling signalcompared to the level of labeling in a non-high grade breast carcinomacontrol in step e) prepared using the same steps as the test sampleindicates a non-high grade breast carcinoma in the test sample that isnon-invasive; and wherein a test sample having a greater level oflabeling signal compared to the level of labeling in a non-high gradebreast carcinoma control in step e) prepared using the same steps as thetest sample indicates a high grade breast carcinoma in the test samplethat is invasive.
 2. The method of claim 1 wherein the detection reagentis an oligonucleotide probe capable of hybridizing with human BC200 RNA.3. The method of claim 1 wherein RT-PCR is utilized to produce adetectable reaction product if human BC200 RNA is present.